Neuroprotective effects of leptin in vivo and in vitro.

The present study explored the efficacy of leptin in protecting against in vivo induction of excitotoxic lesions by the glutamatergic analogue ibotenate injected into the developing mouse brain and against in vitro NMDA-induced cell death in primary neuronal cultures. Ibotenate injected intracerebrally (i.c.) to mice on postnatal day 5 produced transcortical necrosis and white matter cysts. Co-treatment with leptin administered i.c. or i.p. reduced ibotenate-induced cortical lesions and white matter cysts by 50%. in vitro, leptin afforded significant neuroprotection of mouse cortical neurons against NMDA cytotoxicity. The neuroprotective effect of leptin was antagonized both in vivo and in vitro by the Jak2 inhibitor AG490, indicating that it was mediated via the leptin receptor and Jak2 activation. These findings are the first evidence for a role of leptin in neuroprotection.     

Neuroprotective actions in vivo and electrophysiological actions in vitro of 202W92

202W92 (R-(-)-2,4-diamino-6-(fluromethyl)-5-(2,3,5-trichlorophenyl)pyrimidine) is a novel compound in the same chemical series as the antiepileptic drug lamotrigine and the neuroprotective sipatrigine. Here 202W92 was quantitatively assessed as a neuroprotective agent in focal cerebral ischaemia, and as an inhibitor of sodium and calcium channels and of synaptic transmission. In the rat permanent middle cerebral artery occlusion (MCAO) model of acute focal ischaemia, 202W92 reduced infarct volume by 75% in cortex and by 80% in basal ganglia, with ED(50) approximately 2 mg/kg (single i.v. dose, 10 min post-occlusion). In whole-cell current recordings from single cells, 202W92 completely and reversibly inhibited voltage gated sodium channels (IC(50) 3 x 10(-6) M) in rat freshly-isolated cortical neurons and in the GH(3) pituitary cell line. 202W92 also inhibited a nifedipine-sensitive fraction (approximately 35%) of native high-voltage-activated (HVA) calcium current in rat cortical neurons (IC(50) 15 x 10(-6) M) and weakly inhibited low-voltage-activated (LVA) calcium currents of the recombinant alpha1I-mediated T-type (IC(50)>100 x 10(-6) M). The drug inhibited the amplitude and frequency of 4-aminopyridine-evoked glutamatergic excitatory post-synaptic currents (EPSCs). In conclusion, 202W92 is an effective neuroprotective agent when administered post-ischaemia and a potent sodium channel inhibitor in vitro.     

Neuroprotective properties of memantine in different in vitro and in vivo models of excitotoxicity

The pathogenesis of stroke, trauma and chronic degenerative diseases, such as Alzheimer's disease (AD), has been linked to excitotoxic processes due to inappropriate stimulation of the N-methyl-D-aspartate receptor (NMDA-R). Attempts to use potent competitive NMDA-R antagonists as neuroprotectants have shown serious side-effects in patients. As an alternative approach, we were interested in the anti-excitotoxic properties of memantine, a well-tolerated low affinity uncompetitive NMDA-R antagonist presently used as an anti-dementia agent. We explored in a series of models of increasing complexity, whether this voltage-dependent channel blocker had neuroprotective properties at clinically relevant concentrations. As expected, memantine protected neurons in organotypic hippocampal slices or dissociated cultures from direct NMDA-induced excitotoxicity. However, low concentrations of memantine were also effective in neuronal (cortical neurons and cerebellar granule cells) stress models dependent on endogenous glutamate stimulation and mitochondrial stress, i.e. exposure to hypoxia, the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+) or a nitric oxide (NO) donor. Furthermore, memantine reduced lethality and brain damage in vivo in a model of neonatal hypoxia-ischemia (HI). Finally, we investigated functional rescue (neuronal capacity to migrate along radial glia) by memantine in cerebellar microexplant cultures exposed to the indirect excitotoxin 3-nitropropionic acid (3-NP). Potent NMDA-R antagonists, such as (+)MK-801, are known to block neuronal migration in microexplant cultures. Interestingly, memantine significantly restored the number of neurons able to migrate out of the stressed microexplants. These findings suggest that inhibition of the NMDA-R by memantine is sufficient to block excitotoxicity, while still allowing some degree of signalling.     

Neuroprotective effects of minocycline against in vitro and in vivo retinal ganglion cell damage

The purpose of this study was to determine whether minocycline, a semi-synthetic tetracycline derivative, reduces (a) the in vitro neuronal damage occurring after serum deprivation in cultured retinal ganglion cells (RGC-5, a rat ganglion cell line transformed using E1A virus) and/or (b) the in vivo retinal damage induced by N-methyl-D-aspartate (NMDA) intravitreal injection in mice. In addition, we examined minocycline's putative mechanisms of action against oxidative stress and endoplasmic reticulum (ER) stress. In vitro, retinal damage was induced by 24-h serum deprivation, and cell viability was measured by Hoechst 33342 staining or resazurin reduction assay. In cultures of RGC-5 cells maintained in serum-free medium for up to 24 h, the number of cells undergoing cell death was reduced by minocycline (0.2-20 microM). Serum deprivation resulted in increased oxidative stress, as revealed by an increase in the fluorescence intensity for 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), a reactive oxygen species (ROS) indicator. Minocycline at 2 and 20 microM inhibited this ROS production. However, even at 20 microM minocycline did not inhibit the retinal damage induced by tunicamycin (an ER stress inducer). Furthermore, in mice in vivo minocycline at 90 mg/kg intraperitoneally administered 60 min before an NMDA intravitreal injection reduced the NMDA-induced retinal damage. These findings indicate that minocycline has neuroprotective effects against in vitro and in vivo retinal damage, and that an inhibitory effect on ROS production may contribute to the underlying mechanisms.     

Neuroprotective effects of an estratriene analog are estrogen receptor independent in vitro and in vivo

Estrogens are potent neuroprotectants both in vitro and in vivo. In the present study, we compared the potency and efficacy of a non-feminizing estrogen, 2-(1-adamantyl)-4-methylestrone (ZYC-26), with its parent estrogen, estrone, and an expected non-neuroprotective 3-O-methyl analog of (17beta)-2-(1-adamantyl)estradiol (ZYC-23). These estratriene derivatives were tested for their ability to protect in an in vitro lipid peroxidation model, to neuroprotect against oxidative stress in cell culture models, to bind the estrogen receptors (ERalpha and ERbeta), to elicit uterotrophic effects, and to affect brain damage from transient middle cerebral artery occlusion. We observed that in contrast to estrone, neither ZYC-26 nor ZYC-23 bound to either estrogen receptors (ER) and both failed to elicit a uterotrophic response. In vitro, the active estrogen analogue ZYC-26 was more potent that estrogen in its ability to inhibit lipid peroxidation and to protect HT-22 cells from either glutamate or iodoacetic acid (IAA) toxicity. Further, ZYC-26 was as active in preventing brain damage from transient middle cerebral artery occlusion (MCAO) as was estrone. Collectively, these studies suggest that the antioxidant activity, rather than ER binding of non-feminizing estrogens such as ZYC-26, mediates their potent neuroprotective activity. Further, in view of the now known toxicities of chronic feminizing estrogen use in older women, non-feminizing estrogens may be a useful alternative for estrogen-induced brain protection.     

Neuroprotective effects of insulin-like growth factor-binding protein ligand inhibitors in vitro and in vivo.

The role of brain insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in neuroprotection was further investigated using in vitro and in vivo models of cerebral ischemia by assessing the effects of IGF-I, IGF-II, and high affinity IGFBP ligand inhibitors (the peptide [Leu24, 59, 60, Ala31]hIGF-I (IGFBP-LI) and the small molecule NBI-31772 (1-(3,4-dihydroxybenzoyl)-3-hydroxycarbonyl-6, 7-dihydroxyisoquinoline), which pharmacologically displace and elevate endogenous, bioactive IGFs from IGFBPs. Treatment with IGF-I, IGF-II, or IGFBP-LI (2 microg/mL) significantly (P < 0.05) reduced CA1 damage in organotypic hippocampal cultures resulting from 35 minutes of oxygen and glucose deprivation by 71%, 60%, and 40%, respectively. In the subtemporal middle cerebral artery occlusion (MCAO) model of focal ischemia, intracerebroventricular (icv) administration of IGF-I and IGF-II at the time of artery occlusion reduced ischemic brain damage in a dose-dependent manner, with maximum reductions in total infarct size of 37% (P < 0.01) and 38% (P < 0.01), respectively. In this model of MCAO, i.c.v. administration of NBI-31772 at the time of ischemia onset also dose-dependently reduced infarct size, and the highest dose (100 microg) significantly reduced both total (by 40%, P < 0.01) and cortical (by 43%, P < 0.05) infarct volume. In the intraluminal suture MCAO model, administration of NBI-31772 (50 microg i.c.v.) at the time of artery occlusion reduced both cortical infarct volume (by 40%, P < 0.01) and brain swelling (by 24%, P < 0.05), and it was still effective when treatment was delayed up to 3 hours after the induction of ischemia. These results further define the neuroprotective properties of IGFs and IGFBP ligand inhibitors in experimental models of cerebral ischemia.     

Neuroprotective effects of neuropeptide Y-Y2 and Y5 receptor agonists in vitro and in vivo

It is generally assumed that neurodegeneration is connected with glutamatergic hyperactivity, and that neuropeptide Y (NPY) inhibits glutamate release. Some earlier studies indicated that NPY may have neuroprotective effect; however, the results obtained so far are still divergent, and the role of different Y receptors remains unclear. Therefore in the presented study we investigated the neuroprotective potential of NPY and its Y2, Y5 or Y1 receptor (R) ligands against the kainate (KA)-induced excitotoxicity in neuronal cultures in vitro, as well as in vivo after intrahippocampal KA injection and also in an ischemic middle cerebral artery occlusion model after intraventricular injection of Y2R agonist. NPY compounds were applicated 30 min, 1, 3 or 6 h after the start of the exposure to KA, or 30 min after the onset of ischemia. Our results indicate the neuroprotective activity of NPY and its Y2R and Y5R ligands against the kainate-induced excitotoxicity in primary cortical and hippocampal cultures. Importantly, NPY was effective when given as late as 6 h, while Y2R or Y5R agonists 3 h, after starting the exposure to KA. In in vitro studies those protective effects were inhibited by the respective receptor antagonists. Neuroprotection was also observed in vivo after intrahippocampal injection of Y2R and Y5R agonists 30 min or 1 h after KA. No protection was found either in vitro or in vivo after the Y1R agonist. The Y2R agonist also showed neuroprotective activity in the ischemic model. The obtained results indicate that neuropeptide Y produces neuroprotective effect via Y2 and Y5 receptors, and that the compounds may be effective after delayed application.     

雌激素对癫痫持续状态大鼠海马CA3区神经元保护作用的研究

目的研究雌激素对癫癎持续状态大鼠海马CA3区神经元保护作用.方法利用尼氏染色和免疫组化染色技术,观察癫癎发作前后给予雌激素治疗对癫癎发作造成神经元凋亡及Bcl-2、Bax表达的影响.结果癫癎发作前给予雌激素治疗可明显减轻持续癫癎发作造成的神经元缺失并可抑制Bax表达,但对Bcl-2表达无明显影响.结论雌激素对癫癎持续状态导致的海马神经元损伤有保护作用,对凋亡促进基因Bax表达的影响可能参预了其神经保护作用.     

Neuroprotective effect of tiagabine in transient forebrain global ischemia: an in vivo microdialysis,behavioral, and histological study

The neuroprotective effect of tiagabine was investigated in global ischemia in gerbils. Two groups of the animals received 15 mg/kg of tiagabine 30 min before ischemia. In the first group, the temperature was controlled at 37 degrees C from time of injection to 1 h after ischemia. In the second group, the temperature was left uncontrolled to see the hypothermic effect of tiagabine. Microdialysis was performed in CA1 region of hippocampus in half of the animals in each group to assess the levels of glutamate and gamma-amino-butyric acid (GABA). Animal behavior was also tested in 28-day groups in a radial-arm maze. Histology was done 7 and 28 days after ischemia in CA1 region of hippocampus to assess early and delayed effect of drug. A significant suppression of glutamate was noted in both groups (P<0.01). Behavioral results showed that in the temperature-uncontrolled treatment group, animals significantly reduced their working memory errors as compared to the temperature-controlled treatment group. Histology revealed a significant neuroprotection (P<0.001) in the temperature-uncontrolled treatment group. In the temperature-controlled treatment group, however, neuroprotection was insignificant (P>0.05). A third group of animals received the same dose of tiagabine 3 h after ischemia. Temperature was not controlled in this group. The animals were sacrificed after 7 days so no behavior testing was carried out. Histology showed no neuroprotection in this group (P>0.05). These results show that tiagabine offers a significant neuroprotection in global ischemia in gerbils when given 30 min before ischemia but not when given 3 h after ischemia.     

Neuroprotective effects of MK-801 in vivo: selectivity and evidence for delayed degeneration mediated by NMDA receptor activation

The ability of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 to prevent neuronal degeneration in the rat striatum and hippocampus caused by intracerebral injection of excitotoxins has been examined. Excitotoxic damage was assessed after 7 d, using histological and biochemical [choline acetyltransferase (ChAT) glutamate decarboxylase (GAD)] measurements. Systemically administered MK-801 was found to protect against neurodegeneration caused by NMDA (200 nmol) and the naturally occurring NMDA receptor agonist quinolinate (120-600 nmol) but not against that induced by kainate (5 nmol) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; 50 nmol), indicating a selectivity for NMDA receptor-mediated neuronal loss. Neurotoxicity caused by NMDA (200 nmol) or quinolinate (200 nmol) was prevented by MK-801 (1-10 mg/kg, i.p.) administered in a single dose after excitotoxin injection. In the striatum, significant protection of cholinergic neurons (assessed by ChAT measurements) was observed when MK-801 was given up to 5 hr after injection of NMDA or quinolinate, whereas protection of GABAergic neurons (assessed by GAD measurements) was obtained up to 2 hr. The results suggest that GABAergic neurons degenerate more rapidly than cholinergic neurons. The competitive NMDA receptor antagonist 3-[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonate (100 mg/kg, i.p.) gave partial protection of striatal neurons when administered 1 hr after quinolinate injection. In the rat hippocampus, administration of 10 mg/kg MK-801 i.p. 1 hr after quinolinate injection caused almost complete protection of pyramidal and granule neurons, whereas the degeneration of CA3/CA4 pyramidal neurons caused by kainate injection was unaffected. These observations indicate that neurons in rat striatum and hippocampus do not die as an immediate consequence of exposure to high concentrations of NMDA agonists but that a delayed process is involved that requires NMDA receptor activation. In this respect, intracerebral injections of NMDA agonists may mimic the pathological changes that are thought to occur in the brain following periods of cerebral ischemia, where delayed neuronal degeneration occurs.     

Neuroprotective properties and mechanisms of erythropoietin in in vitro and in vivo experimental models for hypoxia/ischemia

Besides its established function in erythropoiesis, erythropoietin (EPO) is currently also appreciated for its neuroprotective effects. The detrimental sequelae of prolonged cerebral hypoxia and ischemia have been shown to attenuate by EPO treatment. After binding to the EPO receptor, EPO is capable of initiating a cascade of events which--via different pathways--may lead to neuroprotection. The circumstances that determine which specific signalling route(s) are activated by EPO are largely unknown. We aim to provide the reader with a timely overview on the use of EPO in models of stroke and hypoxia-ischemia and to discuss the molecular events that underlie its neuroprotection.     

Neuroprotective Role of Antidiabetic Drug Metformin Against Apoptotic Cell Death in Primary Cortical Neurons

Oxidative damage has been reported to be involved in the pathogenesis of diabetic neuropathy and neurodegenerative diseases. Recent evidence suggests that the antidiabetic drug metformin prevents oxidative stress-related cellular death in non-neuronal cell lines. In this report, we point to the direct neuroprotective effect of metformin, using the etoposide-induced cell death model. The exposure of intact primary neurons to this cytotoxic insult induced permeability transition pore (PTP) opening, the dissipation of mitochondrial membrane potential (ΔΨm), cytochrome c release, and subsequent death. More importantly, metformin, together with the PTP classical inhibitor cyclosporin A (CsA), strongly mitigated the activation of this apoptotic cascade. Furthermore, the general antioxidant N-acetyl-l-cysteine also prevented etoposide-promoted neuronal death. In addition, metformin was shown to delay CsA-sensitive PTP opening in permeabilized neurons, as triggered by a calcium overload, probably through its mild inhibitory effect on the respiratory chain complex I. We conclude that (1) etoposide-induced neuronal death is partly attributable to PTP opening and the disruption of ΔΨm, in association with the emergence of oxidative stress, and (2) metformin inhibits this PTP opening-driven commitment to death. We thus propose that metformin, beyond its antihyperglycemic role, can also function as a new therapeutic tool for diabetes-associated neurodegenerative disorders. El-Mir and Detaille have contributed equally to this work.     

Comparative Neuroprotective Effects of Rasagiline and Aminoindan with Selegiline on Dexamethasone-Induced Brain Cell Apoptosis

Stress can affect the brain and lead to depression; however, the molecular pathogenesis is unclear. An association between stress and stress-induced hypersecretion of glucocorticoids occurs during stress. Dexamethasone (a synthetic glucocorticoid steroid) has been reported to induce apoptosis and increase the activity of monoamine oxidase (MAO) (Youdim et al. 1989). MAO is an enzyme for the degradation of aminergic neurotransmitters; dopamine, noradrenaline and serotonin and dietary amines and MAO inhibitors are classical antidepressant drugs. In this study, we have compared the ability of rasagiline (Azilect) and its main metabolite, R-aminoindan with selegiline (Deprenyl) in prevention of dexamethasone-induced brain cell death employing human neuroblastoma SH-SY5Y cells and glioblastoma 1242-MG cells. Dexamethasone reduced cell viability as measured by MTT test, but rasagiline, selegiline, and 1-R-aminoindan could significantly prevent dexamethasone-induced brain cell death. Among three drugs, rasagiline had the highest neuroprotective effect. Furthermore, the inhibitory effects of these drugs on MAO B catalytic activity and on apoptotic DNA damage (TUNEL staining) were examined. Rasagiline exhibited highest inhibition on MAO B enzymatic activity and prevention on DNA damage as compared to selegiline and 1-R-aminoindan. In summary, the greater neuroprotective effect of rasagiline may be associated with the combination of the parent drug and its metabolite 1-R-aminoindan.